The overall goal of these studies is to test the ability of self-cleaving multimeric ribozyme constructs to ameliorate phenotypic features of osteogenesis imperfecta (OI). We propose that the dominant-negative effect of "structural" or non-excluded type I collagen mutations associated with the most severe forms of OI must be taken into account in all strategies aimed at correcting the collagen defects in those patients, and that elimination of mutant alleles must be highly efficient, since even low levels of mutant allelic products can have a major deleterious effect. Experiments described in this proposal are designed to advance our development of ribozyme genes that effectively target type I collagen mutations typically observed in severe OI patients. In particular, self-cleaving multimeric ribozyme expressing genes will be designed and tested. These may provide sufficiently high ribozyme delivery to target cells so that a greater degree of allelic selectivity can be designed into ribozyme subunits that would ultimately be targeted against heterozygous single nucleotide substitutions. Experiments described in this proposal include: (1) design and testing of self-cleaving multimeric hammerhead ribozyme(s) that selectively cleave adjacent to a unique deletion junction in the human proa1(I) collagen minigene pMG155. Preliminary testing of the ribozymes' efficiency will be performed by RNase protection in vitro. Target constructs will be stably expressed in MC3T3-E1 cells for in cellulo ribozyme testing. Cleavage efficacy and reversal of a well-defined biochemical phenotype in minigene-expressing MC3T3-E1 cells will be demonstrated by Northern and Western blot analysis, pulse-chase labeling of procollagens, and measurement of several markers of cellular differentiation. Transgenic mice that express either pMG155 or the most optimal multimeric ribozyme expression gene will be mated, and the phenotypes of doubly transgenic mice will be compared to those of mice expressing only the target sequence or the ribozyme; (2) similar in vitro, in cellulo, and in vivo testing will be performed on multimeric ribozymes, that target glycine mutations created in modified versions pMG155 containing an in-frame, 270 bp eDNA segment from the 3' end of the triple helical domain of the murine colla2 gene; and (3) we will test possible additive therapeutic benefits of targeting a potential downstream mediator of the OI dominant negative phenotype, osteocalcin, in mice transgenic for a collagen minigene and optimal ribozyme construct targeting both the minigene and osteocalcin.